A glycoprotein is a protein containing carbohydrate residue(s) in addition to the polypeptide chain where the protein/sugar complex is formed by covalent bonding with sugar by glycosylation of the amino acid residue of the protein during the post-translational modification of the protein. It is known that the glycosylation of a protein is catalyzed by N-acetylglucosaminyltransferase and that the complex carbohydrates of a glycoprotein are synthesized in which monosaccharides are sequentially transferred to the amino acid residues (such as serine, threonine and asparagine) of the protein resulting in the generation of the glycoprotein.
Glycosylation of a protein is divided into N-linked glycosylation, which occurs through the asparagine side-chain of a glycosylation consensus sequence consisting of amino acid-serine-threonine (NXS/T) excluding asparagine-proline during the protein synthesis, and O-linked glycosylation which occurs through a hydroxyl group of serine or threonine residues. In addition, glycans that are commonly found in glycoprotein include glucose (Glc), galactose (Gal), mannose (Man), fucose (Fuc), N-acetylgalactosamine (GalNAc), N-acetylglucosamine (GlcNAc) and N-acetylneuraminic acid (NeuNAc) (Frank Kjeldsen, et al. Anal. Chem. 2003, 75, 2355-2361).
It is known that the sugar chains of many kinds of glycoproteins present on the cell surface are involved in many of the basic phenomena of multicellular processes, including intercellular recognition and adhesion in the modification, development and differentiation processes including the induction of differentiation of immune and neural cells, infection of host cells by bacteria and viruses, adhesion of toxins to cells, cell carcinogenesis and cancer metastasis, and the like.
In addition, the difference in the glycosylation of a glycoprotein can be an important clinical index to distinguish the effect of a targeted therapeutic agent or resistance to the therapeutic agent. For example, according to Kim J G et al. (2012). “Heterodimerization of glycosylated insulin-like growth factor-1 receptors and insulin receptors in cancer cells sensitive to anti-IGF1R antibody”; PLoS One; 7(3):e33322, it is known that resistance to an anticancer therapeutic agent in liver cancer is associated with a change in N-linked glycosylation.
Thus, studies on the difference in the glycosylation of a glycoprotein make it possible to predict the prognosis of disease and obtain information for therapeutic responses to predict the effect of a target therapeutic agent.
Accordingly, antibodies that target the sugar chains of a glycoprotein have recently been studied and developed. For example, a method for developing an antibody that targets sugar chains has been proposed, which comprises immobilizing a chemically synthesized glycan onto a plate and screening an antibody that binds specifically to the glycan (Blixt, O., S. Head, et al. (2004). “Printed covalent glycan array for ligand profiling of diverse glycan binding proteins.” Proc Natl Acad Sci USA 101(49): 17033-17038). However, the antibody selected by the above method targets the chemically synthesized glycan, and thus the actual application of the antibody to sugar chains bound to proteins such as glycoprotein in vivo was limited.
Accordingly, the inventors in the present invention have invented a method for producing a novel antibody, which comprises proteolyzing a glycoprotein obtained from a biological sample to extract short-length glycopeptide(s) containing a glycan and applying a phage antibody display method to the glycopeptide(s), thereby completing the present disclosure.